Markers of unstable atherosclerotic plaques

ABSTRACT

The present invention relates to (the use of) polynucleotides differentially expressed in ruptured and stable atherosclerotic plaques as marker for atherosclerosis, prevention and treatment of atherosclerosis disorders.

[0001] The invention relates to the use of polynucleotidesdifferentially expressed in ruptured and stable atherosclerotic plaquesas marker for atherosclerosis, a method for determining the presence ofsaid polynucleotides in a sample, a method for determining the presenceof amino acids encoded by said polynucleotides in a sample, a diagnosticprocess wherein the expression level of said polynucleotides isdetermined, a diagnostic process wherein a sample is analyzed for thepresence of said amino acid sequences as well as to a newly identifiedpolynucleotide and the amino acid sequence encoded by thatpolynucleotide.

[0002] Atherosclerosis is a major problem in the western world and isthe main cause of cardiovascular disease and deaths. It is a systemicchronic progressive disease affecting all major arteries.Atherosclerotic cardiovascular disease comprises a number ofpathological conditions, such as acute coronary syndromes like ischemic(or coronary) heart disease (MID), stroke, and peripheral vasculardisease.

[0003] Although it may take at least 30 to 40 years to become clinicallymanifest, one may conclude that atherosclerosis—though not always insevere forms—affects all adult individuals in the western world.

[0004] Endothelial dysfunction is one of the initiating events ofchronic atherosclerosis, a slowly growing atherosclerotic plaque thatencroaches the lumen and reduces the lumen. Endothelial dysfunction isassociated with an apparent decrease in the synthesis of the vasodilatornitric oxide (NO). The subsequent development of the atheroscleroticlesion progresses through five stages, from early lesion to stenotic orthrombogenic and occlusive plaque. The different plaque types aredefined by histological criteria (Stary, et al., Circulation 1999; 92:1355-1374). Most clinical atherosclerotic symptoms (about two-thirds ofcoronary occlusions) are related to the transition of a stableatherosclerotic plaque into a ruptured atherosclerotic plaque. Ruptureof unstable atherosclerotic plaques is characterised by a suddenactivation of the clotting system, leading to a sudden occlusion of thelumen (thrombosis) (Libby, Circulation 1995; 91: 2844-2850; Dollery, etal., Circ. Res. 1995; 77: 863-868; Davies, Circulation 1996; 94:2013-2020). Treatments that increase or maintain plaque stability maytherefore for instance decrease the risk of coronary syndromes inpatients with IHD, or decrease the risk of other clinical eventsassociated with cardiovascular disease.

[0005] Patient groups in which atherosclerosis is the major cause ofdisease include patients with a myocardial infarction, angina pectoris,unstable angina, cerebral ischemia and infarction, dementia, andperipheral and intestinal ischemia. Patients at high risk for developing(premature) symptoms of atherosclerosis are those that have high serumcholesterol levels (in low density lipoprotein (LDL) or very low densitylipoprotein (VLDL) particles), or high levels of triglycerides,lipoprotein (a), or fibrinogen, or those people that smoke, havehypertension, have diabete mellitus, or have familial (genetic)disorders in their lipoprotein metabolism, such as familial combinedhyperlipidemia. All these patients may benefit from the utility ofunstable plaque specific diagnostics/therapeutics.

[0006] Although it is known that a ruptured plaque causes the majorityof clinical symptoms of atherosclerotic cardiovascular disease (Ross R.,N Engl J Med 1999; 340:115-26; Libby P., J Intern Med 2000; 247:349-58;Zaman A. G., et al., Atherosclerosis 2000; 149:251-66.), it is notunravelled yet which factors and molecular mechanisms are responsiblefor the transition of a stable plaque into a ruptured plaque.

[0007] The morphology of ruptured plaques is well described (Stary H.C., et al., Arterioscler Thromb Vasc Biol 1995; 15:1512-31; Virmani R,et al., Arterioscler Thromb Vasc Biol 2000;20:1262-75), however,specific markers to identify ruptured plaques or plaques prone torupture in vivo are not available (Kullo I. J., et al., Ann Intern Med1998; 129:1050-60).

[0008] In an attempt to shed more light on the possible molecularmechanisms involved in the onset and progression of atherosclerosis,several studies compared gene expression of activated human umbilicalvein endothelial cells and vascular smooth muscle cells to non-activatedcells (Lu K. P., et al., Biochem Biophys Res Commun 1998; 253:828-33;Sato N., et al., J Biochem (Tokyo) 1998; 123:1119-26; De Waard V, etal., Gene 1999; 226:1-8; Horrevoets A. J., et al., Blood 1999;93:3418-3431; De Vries C. J., et al., J Biol Chem 2000:275:31:23939-47). These studies in cell lines, revealed differentialregulation of genes involved in leukocyte trafficking, cell cyclecontrol and apoptosis. However, although cell lines do provide areproducible source of RNA, it remains to be determined whether geneexpression in vitro mimics gene expression in vivo. Others (Hiltunen M.O., Curr Opin Lipidol 1999; 10:515-9) used whole mount humanatherosclerotic plaques to study differences in gene expression betweenfatty streaks and advanced lesions. They however, did not validate theirfindings on a large panel of individual patients and did not study thelocalization of differentially expressed genes.

[0009] The present invention relates to the use of a polynucleotidedifferentially expressed in ruptured (unstable) and stableatherosclerotic plaques as a marker for atherosclerosis wherein thepolynucleotide is encoding an amino acid sequence selected from SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO: 5. It has now been found that saidpolynucleotides are either upregulated or downregulated in unstableatherosclerotic plaques.

[0010] Polynucleotides comprising SEQ ID NO:3 and SEQ ID NO:5 arealready known in the art (e.g. from WO 9946380 and Accession Number AK000362, respectively) as membrane spanning protein and human sortingnexin, respectively. There is no indication in the art, that these genesmight be upregulated or downregulated in unstable plaque tissue.

[0011] A particular preferred embodiment of the invention relates to anovel polynucleotide that is highly expressed in unstable, rupturedatherosclerotic lesions. More specifically, the present inventionprovides for an isolated polynucleotide encoding the amino acid sequenceSEQ ID:1. The term isolated denotes that the polynucleotide has beenremoved from its natural environment and is thus in a form suitable foruse within genetically engineered protein production systems.

[0012] The invention also includes a polynucleotide comprising the DNAsequence which is indicated in SEQ ID NO: 2. In particular preferred isa polynucleotide comprising the complete coding DNA sequence of thenucleotides 1169-2587 of SEQ ID NO:2. Furthermore, to accommodate codonvariability, the invention also includes sequences coding for the sameamino acid sequences as the sequences disclosed herein (SEQ ID NO:1).Also portions of the coding sequences coding for individual domains ofthe expressed protein are part of the invention as well as allelic andspecies variations thereof Sometimes, a gene is expressed in a certaintissue as a splicing variant, resulting in the inclusion of anadditional exon sequence, or the exclusion of an exon. Also a partialexon sequence may be included or excluded. A gene may also betranscribed from alternative promotors that are located at differentpositions within a gene, resulting in transcripts with different 5′ends. Transcription may also terminate at different sites, resulting indifferent 3′ ends of the transcript. These sequences as well as theproteins encoded by these sequences all are expected to perform the sameor similar functions and form also part of the invention.

[0013] The sequence information as provided herein should not be sonarrowly construed as to require inclusion of erroneously identifiedbases. The specific sequence disclosed herein can be readily used toisolate the complete genes which in turn can easily be subjected tofurther sequence analyses thereby identifying sequencing errors.

[0014] The polynucleotides of this invention, which are differentiallyexpressed in ruptured and stable atherosclerotic plaques, or theproteins encoded, are important tools for diagnostics and therapeutics.

[0015] As a diagnostic tool a differentially expressed gene can be usedas a marker for unstable plaques in an individual, where the expressionlevels of the gene in tissue samples are determined. Further, it may beused to identify other sites of plaque instability in a patient thatshows clinical symptoms of an unstable plaque of an artery, like theiliac artery, leading to peripheral ischernia. Since the long termprognosis of those patients is not determined by the success rate of theperipheral interventions, but by the occurrence of a myocardial orcerebral infarction, the correct diagnosis of all sites of plaqueinstability is of utmost importance. Diagnostic techniques such asimaging techniques, e.g. scintigraphy, may be applied, in which theradiolabeled unstable plaque specific gene is used as the target.Alternatively, the polynucleotides of this invention representing anunstable plaque specific gene, or the proteins encoded, may be used asserum/plasma markers, which may also be used to screen patients at riskfor plaque instability or to evaluate the effects of other treatments.Further, the (novel) unstable plaque specific polynucleotides of thisinvention, or the encoded proteins or antibodies against the proteins,may be used to target other therapeutics to an unstable plaque.

[0016] Therefore, another aspect of the present invention is a methodfor determining the presence of a polynucleotide in a sample comprising:obtaining polynucleotides from an individual (e.g. by taking tissuesamples, blood samples and the like, using (clinical) methods well knownin the art for such purpose) and determining whether a polynucleotideencoding an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3or SEQ ID NO: 5 is present. Any method for detection of(poly)nucleotides known in the art for such purpose is includedherewith. For example, nucleotide elongation methods/amplificationmethods may be considered, but also, such method may comprise the stepsof: hybridizing to a sample a probe specific for a polynucleotideencoding an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3or SEQ ID NO: 5 under conditions effective for said probe to hybridizespecifically to said polynucleotide and determining the hybridization ofsaid probe to polynucleotides in said sample. The term “specific” inthis respect means that the majority of hybridization takes place with apolynucleotide of this invention. Preferably, said probe comprises atleast 25 of the nucleotides of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.More preferred, the probe comprises 50, and in particular preferred morethan 100, nucleotides of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. Mostpreferred, the probe consists of a polynucleotide of nucleotidesselected from the nucleotides 1169 to 2587 of SEQ ID NO:2. Appropriatestringency conditions which promote DNA hybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by a washof 2.0×SSC at 50° C., are known to those skilled in the art or can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. For example, the salt concentration in the washstep can be selected from low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.

[0017] A further aspect of the present invention is a method fordetecting in a sample a protein with amino acid sequence selected fromSEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO: 5, said method comprising:incubating with a sample a reagent that binds specifically to saidprotein (e.g. an antibody) under conditions effective for specificbinding and determining the binding of said reagent to said protein insaid sample.

[0018] In addition, a diagnostic process is an embodiment of the presentinvention comprising: determining the difference in expression level ofa polynucleotide encoding an amino acid sequence selected from SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO: 5 in a sample derived from a host whencompared to a known standard (e.g. using the above mentioned methods).Said known standard relates to healthy tissues and stable plaquematerial of healthy individuals. When the expression level of saidpolynucleotide of the present invention is upregulated or downregulatedwhen compared to that standard, the host (usually a human being) fromwhich the sample was derived, is at risk for atherosclerosis. Further,another aspect of this invention is a diagnostic process comprising:analyzing for the presence of the protein with amino acid sequenceselected from SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO: 5 in a samplederived from a host (the methods for which analysis are well known inthe art e.g. using the above mentioned methods). When the amount of theprotein is higher or lower than the amount present in healthy tissueand/or stable plaque material, the host (usually a human being) fromwhich the sample was derived, is at risk for atherosclerosis.

[0019] As a therapeutic tool, modulation—either directly orindirectly—of the expression of a polynucleotide encoding an amino acidsequence selected from SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO: 5, canincrease plaque stability and thus inhibit the progression ofatherosclerotic cardiovascular disease. For example, blocking antibodiesand/or antagonists against an unstable plaque specific gene may be usedto prevent the transition of a stable to an unstable plaque or toreverse an unstable plaque into a stable plaque. Further, the regulationof expression of the corresponding protein and the amount of the proteinpresent in bodily tissues and fluids may be affected by regulation ofthe promotor of the gene or by the use of specifically synthesizedantisense RNA for gene therapy.

[0020] The DNA according to the invention may be obtained from cDNAusing suitable probes derived from SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6. Alternatively, the coding sequence might be genomic DNA, orprepared using DNA synthesis techniques. The polynucleotide may also bein the form of RNA. If the polynucleotide is DNA, it may be in singlestranded or double stranded form. The single strand might be the codingstrand or the non-coding (anti-sense) strand.

[0021] The present invention further relates to polynucleotides whichhave at least 70%, preferably 80%, more preferably 90%, even morepreferred 95%, and highly preferably 98% and most preferred at least 99%identity with the entire DNA sequence of the nucleotides 1169-2587 ofSEQ ID NO:2. Such polynucleotides encode polypeptides which retain thesame biological function or activity as the natural, mature protein.Alternatively, also fragments of the above mentioned polynucleotideswhich code for domains of the protein which still are capable of bindingto substrates are embodied in the invention.

[0022] The percentage of identity between two sequences can bedetermined with programs such as DNAMAN (Lynnon Biosoft, version 3.2).Using this program two sequences can be aligned using the optimalalignment algorithm of Smith and Waterman (1981, J. Mol. Biol,147:195-197). After alignment of the two sequences the percentageidentity can be calculated by dividing the number of identicalnucleotides between the two sequences by the length of the alignedsequences minus the length of all gaps.

[0023] The present invention further relates to (the use of)polynucleotides having slight variations or having polymorphic sites.Polynucleotides having slight variations encode polypeptides whichretain the same biological function or activity as the natural, matureprotein.

[0024] The sequence of the newly identified polynucleotide of thepresent invention, SEQ ID NO:2, and the sequences SEQ ID NO:4 or SEQ IDNO:6 may also be used in the preparation of vector molecules for theexpression of the encoded protein in suitable host cells. A wide varietyof host cell and cloning vehicle combinations may be usefully employedin cloning the nucleic acid sequences coding for the proteins of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 or parts thereof For example, usefulcloning vehicles may include chromosomal, non-chromosomal and syntheticDNA sequences such as various known bacterial plasmids and wider hostrange plasmids and vectors derived from combinations of plasmids andphage or virus DNA.

[0025] Vehicles for use in expression of the polynucleotides of thepresent invention or a part thereof comprising a functional domain willfurther comprise control sequences operably linked to the nucleic acidsequence coding for the protein. Such control sequences generallycomprise a promoter sequence and sequences which regulate and/or enhanceexpression levels. Of course control and other sequences can varydepending on the host cell selected.

[0026] Suitable expression vectors are for example bacterial or yeastplasmids, wide host range plasmids and vectors derived from combinationsof plasmid and phage or virus DNA. Vectors derived from chromosomal DNAare also included. Furthermore an origin of replication and/or adominant selection marker can be present in the vector according to theinvention. The vectors according to the invention are suitable fortransforming a host cell. Recombinant expression vectors comprising DNAof the invention as well as cells transformed with said DNA or saidexpression vector also form part of the present invention. Suitable hostcells according to the invention are bacterial host cells, yeast andother fungi, insect, plant or animal host cells such as Chinese HamsterOvary cells or monkey cells or human cell lines. Thus, a host cell whichcomprises DNA or expression vector according to the invention is alsowithin the scope of the invention. The engineered host cells can becultured in conventional nutrient media which can be modified e.g. forappropriate selection, amplification or induction of transcription. Theculture conditions such as temperature, pH, nutrients etc. are wellknown to those ordinary skilled in the art.

[0027] The techniques for the preparation of DNA or the vector accordingto the invention as well as the transformation or transfection of a hostcell with said DNA or vector are standard and well known in the art, seefor instance Sambrook et al., Molecular Cloning: A laboratory Manual.2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

[0028] In another aspect of the invention, there is provided for aprotein comprising the amino acid sequence encoded by the abovedescribed DNA molecules. Preferably, the protein according to theinvention comprises an amino acid sequence shown in SEQ ID NO:1. Alsopart of the invention are proteins resulting from post translationalprocessing, which proteins are encoded by the polynucleotide of thisinvention.

[0029] Also functional equivalents, that is proteins homologous to aminoacid sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or parts thereofhaving variations of the sequence while still maintaining functionalcharacteristics, are included in the invention.

[0030] The variations that can occur in a sequence may be demonstratedby (an) amino acid difference(s) in the overall sequence or bydeletions, substitutions, insertions, inversions or additions of (an)amino acid(s) in said sequence. Amino acid substitutions that areexpected not to essentially alter biological and immunologicalactivities, have been described. Amino acid replacements between relatedamino acids or replacements which have occurred frequently in evolutionare, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see DayhofM. D., Atlas of protein sequence and structure, Nat. Biomed. Res.Found., Washington D.C., 1978, vol. 5, suppl. 3). Based on thisinformation Lipman and Pearson developed a method for rapid andsensitive protein comparison (Science, 1985, 227, 1435-1441) anddetermining the functional similarity between homologous polypeptides.It will be clear that also polynucleotides coding for such variants arepart of the invention.

[0031] The polypeptides according to the present invention also includepolypeptides comprising SEQ ID NO:1, but further polypeptides with aidentity of at least 70%, preferably 80%, more preferably 90%, and evenmore preferred 95%. Also portions of such polypeptides still capable ofconferring biological effects are included. Especially portions whichstill bind to targets form part of the invention. Such portions may befunctional per se, e.g. in solubilized form or they might be linked toother polypeptides, either by known biotechnological ways or by chemicalsynthesis, to obtain chimeric proteins. Such proteins might be useful astherapeutic agent.

[0032] The proteins according to the invention can be recovered andpurified from recombinant cell cultures by common biochemicalpurification methods (as decribed in Havelaar et al, J. Biol. Chem. 273,34568-34574 (1998)) including ammonium sulfate precipitation,extraction, chromatography such as hydrophobic interactionchromatography, cation or anion exchange chromatography or affinitychromatography and high performance liquid chromatography. If necessary,also protein refolding steps can be included. Alternatively the proteincan be expressed and purified as a fusion protein containing (“tags”)which can be used for affinity purification.

[0033] The proteins according to the present invention may be used forthe in vitro or in vivo identification of novel targets or analogues.For this purpose e.g. binding studies may be performed with cellstransformed with DNA according to the invention or an expression vectorcomprising DNA according to the invention, said cells expressing anunstable plaque specific polynucleotide according to the invention.

[0034] Alternatively also the (newly identified) polynucleotidesaccording to the invention as well as the target-binding domain thereofmay be used in an assay for the identification of functional targets oranalogues for the gene.

[0035] Using such an assay compounds can be identified that prevent thetransition of a stable to an unstable plaque or reverse that prevent thetransition of a stable to an unstable plaque or reverse an unstableplaque into a stable plaque an unstable plaque into a stable plaque, andthus inhibit the progression of atherosclerotic disease.

[0036] Thus, the present invention provides for a method for identifyingcompounds that prevent the transition of a stable to an unstable plaqueor reverse an unstable plaque into a stable plaque. The method comprisesthe steps of

[0037] a) introducing into a suitable host cell a nucleotide encodingthe amino acid selected from the sequences of SEQ ID NO:1, SEQ ID NO:3and SEQ ID NO:5;

[0038] b) culturing the host cells under conditions to allow expressionof the introduced DNA sequence;

[0039] c) bringing the host cell of step b, or products thereof, intocontact with compounds potentially effecting the function of theexpressed protein with SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO: 5;

[0040] d) determining the effect of such compounds on the function ofthe expressed protein.

[0041] The present invention thus provides for a quick and economicmethod to screen for therapeutic agents for the prevention and/ortreatment of cardiovascular diseases related to the transition of astable to an unstable plaque.

[0042] The invention also provides for a method for the formulation of apharmaceutical composition comprising mixing modulator compoundsidentified according to the above procedure with a pharmaceuticallyacceptable carrier.

[0043] Pharmaceutical acceptable carriers are well known to thoseskilled in the art and include, for example, sterile saline, lactose,sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil,olive oil, sesame oil and water.

[0044] Furthermore the pharmaceutical composition according to theinvention may comprise one or more stabilizers such as, for example,carbohydrates including sorbitol, mannitol, starch, sucrosedextrin andglucose, proteins such as albumin or casein, and buffers like alkalinephosphates. Methods for making preparations and intravenous admixturesare disclosed in Remingtons's Pharmaceutical Sciences, pp. 1463-1497(16th ed. 1980, Mack Publ. Co of Easton, Pa., USA).

[0045] Thus, the modulator compounds identified by using apolynucleotide according to the invention are useful in the preparationof a pharmaceutical. The pharmaceutical is to be used in atheroscleroticdisorders.

[0046] Also within the scope of the invention are antibodies, especiallymonoclonal antibodies raised against a protein according to theinvention. Such antibodies may be used both therapeutically anddiagnostically. The antibodies can be prepared according to methodsknown in the art e.g. as described in EP488470.

[0047] The invention is further explained by reference to the followingillustrative Examples.

LEGEND TO THE FIGURES

[0048]FIG. 1. Inverse Northern Dot Blot analysis of cDNA clonesgenerated by SSH. Two identical Dot Blots were made by transfer of PCRproducts to nylon membranes. The Dot Blots were hybridized to A) a³²P-labeled cDNA pool of 3 different stable plaques and B) a ³²P-labeledcDNA pool of 3 different ruptured plaques as described in the Examples.The position of SEQ ID NO:2, a clone upregulated in ruptured plaques, isD6.

[0049]FIG. 2. RT-PCR analysis of the newly identified polynucleotide ofSEQ ID NO:2, differentially expressed in ruptured or stable humanatherosclerotic plaques. The figure shows expression in 10 differentstable plaques (left panel) and 10 different ruptured plaques (rightpanel).

[0050]FIG. 3. Inverse Northern Dot Blot analysis of cDNA clonesgenerated by SSH. Two identical Dot Blots were made by transfer of PCRproducts to nylon membranes. The Dot Blots were hybridized to A) a³²P-labeled cDNA pool of 3 different stable plaques and B) a ³²P-labeledcDNA pool of 3 different ruptured plaques as described in the Examples.The position of SEQ ID NO:6, a clone downregulated in ruptured plaques,is C3.

[0051]FIG. 4. Expression of SSH6 in the vascular wall. RT-PCR on mRNAisolated from veins and arteries with atherosclerotic lesions in variousstages.

[0052]FIG. 5. Tissue distribution of the ubiqutiously expressed SSH 6mRNA. Hybridization of a human multiple tissue array with the ³³P-dCTPlabeled cDNA seq of the nucleotides 905-1341 of SEQ ID NO:2. Aschematical representation of the various tissues and cell lines isdepicted in the lower panel.

[0053]FIG. 6. Tissue distribution of SSH6v mRNA. Hybridization of thehuman multiple tissue array with 33 P-dCTP labeled exon 3 (seeschematical representation in the lower panel of FIG. 5 for blotcomposition). The lower panel shows hybridization with VSMC derived RNAand exon 3 containing plasmid DNA as a positive control.

[0054]FIG. 7. Schematic representation of the GST-SSH6 fusion protein.The 302 C-terminal AA deduced from the putative open reading of SSH6 arefused to the C-terminal end of glutathion S-transferase (GST).

[0055]FIG. 8. SSH6 protein expression. Western blot analysis of humanatherosclerotic plaques, human plasma and several human tissue lysatesand cell lines using the SSH6 specific SSH6-scFv. Lane 1: smooth musclecell lysate, lane 2: human aorta, lane 3: LS174T cells, lane 4: LLCcells, lane 5: CaCo cells, lane 6: COS cells, lane 7: marker, lane 8:ruptured atherosclerotic plaque, lane 9: HUVEC cells, lane 10: OVCARcells, lane 11: human plasma.

EXAMPLES

[0056] General

[0057] The technique of suppression subtractive hybridization (SSH)(Diatchenko L, et al., Proc Natl Acad Sci USA 1996; 93:6025-30) wasapplied, using whole mount specimens, for the identification of thepolynucleotide of the present invention which is differentiallyexpressed in whole mount human stable and ruptured plaques.

[0058] To obtain plaque type specific genes, the plaques included in the2 pools (ruptured and stable plaques) were morphologically diverse withrespect to the presence of a lipid core, calcium deposition and theamounts of inflamnnatory cells. Furthermore, the SSH procedure wasperformed on pools of 3 advanced stable lesions (type IV and V) and 3ruptured lesions (type VI), to circumvent patient based differences. Toselect for genes with larger differences in expression the SSH wasperformed with a 4-fold excess of driver. The SSH procedure yielded acDNA library, enriched with clones upregulated in ruptured plaques.Differential expression of a number of randomly chosen clones wasvalidated by Inverse Northern Dot Blot (INDB) analysis. Sequencesshowing an at least 2-fold difference in expression were sequenced. Tovalidate the reproducibility of expression of these sequences, RT-PCRanalysis was performed on a larger series of individual ruptured (n=10)and individual stable (n=10) plaques, which showed a strikingconsistency of expression for the polynucleotide of the presentinvention, present in 8 ruptured and 2 stable plaques.

[0059] The differential expression pattern of the polynucleotide of thepresent invention suggests a potential role for this gene in plaquerupture.

[0060] Tissue Sampling and RNA Isolation

[0061] Plaques were obtained from patients undergoing vascular surgery(Department of General Surgery, Academic Hospital Maastricht). Patientcharacteristics are summarized in Table 1. Immediately after resection,the atherosclerotic specimen was divided into parallel parts of 5 mm forRNA isolation and histological analysis. Tissue destined for RNAisolation was immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated using the guanidine isothiocyanate/CsCl method(Chomczynski P., et al., Anal Biochem 1987;162:156-9). Specimens forhistological analysis were fixed in 10% phosphate buffered formalin (pH7.4), routinely processed and embedded in paraffin. Sections were cut,stained with heamatoxylin and eosin and classified according to themorphological criteria of the American Heart Association (Stary H. C.,et al., Arterioscler Thromb Vasc Biol 1995;15:1512-31). Only advancedatherosclerotic plaques were included in the study. Type IV and Vlesions were defined as stable plaques and type VI lesions were definedas ruptured plaques.

Example 1

[0062] Suppression Subtractive Hybridization

[0063] The SSH procedure was performed using the PCR-Select cDNASubtraction Kit (Clontech) essentially according to the protocol of themanufacturer, with minor adjustments. Briefly, total RNA was isolatedfrom whole mount plaques of 6, age matched, male patients undergoingperipheral vascular surgery (Table 1). To correct for patient baseddifferences in gene expression, 2 pools of total RNA were generated.Pool 1 contained 1 μg of total RNA derived from 3 ruptured plaques of 3individual patients. Pool 2 contained 1 μg of total RNA derived from 3stable plaques of 3 individual patients. The SMART™ PCR cDNA SynthesisKit (Clontech) was used for the preparation and amplification of doublestranded cDNA. In the forward reaction, genes upregulated in rupturedplaques were isolated. RsaI digested tester cDNA was ligated to twodifferent adaptors and hybridized to a 4-fold excess of driver cDNA toenrich for differentially expressed genes. Differentially expressedgenes were amplified by 2 rounds of PCR. The resulting fragments weregel purified, cloned into the pGEMT-easy vector (Promega) andsubsequently transformed to highly competent E. coli JM109 cells(Promega). The thus constructed (forward subtracted) library contained anumber of clones upregulated in ruptured atherosclerotic plaques.

Example 2

[0064] Analysis of Subtracted cDNA Libraries

[0065] Inverse Northern Dot Blotting (INDB)

[0066] Differential expression of the sequences of the SSH library wasverified by a second independent method, the INDB analysis. Sequences ofthe library were randomly chosen and screened for expression in rupturedand stable plaques. This resulted in the identification of a clone thatwas uniquely expressed in ruptured plaques (FIG. 1).

[0067] Procedure: Inserts were amplified by PCR using the T7(5′-TAATACGACTCACTATAGGG-3′, SEQ ID NO:7) and SP6(5′-ATTTAGGTGACACTATA-3′, SEQ ID NO: 8) primers under standardconditions. Briefly, 10 μl of PCR product was diluted in 200 μg 6×SSC,heated to 95° C. and quenched on ice. Two identical dot blots were madeby transferring 100 μl of the sample to a nylon membrane (Nytran;Schleicher & Schuell) using a 96-wells BioRad Dot Blot apparatus and theDNA was subsequently crosslinked by UV irradiation. The filters werehybridized at high stringency with ³²P-labeled (High Prime, BoehringerMannheim) SMART™ cDNA of either stable or ruptured plaques usingstandard procedures. Hybridization signals were normalized usingRNA-polymerase II and genomic DNA signals. Quantitative analysis wasperformed by phosphor image analysis.

Example 3

[0068] Sequencing

[0069] The differentially expressed polynucleotide of Example 2 wassequenced using the Thermo Sequenase fluorescent labelled primer (M13reverse 5′-TTTCACACAGGAAACAGGAAACAGCTATGAC-3′, SEQ ID NO: 9, M13 forward5′-CGCCAGGGTTTTCCCAGTCAC GAC-3′, SEQ ID NO: 10) cycle sequencing kit(Amersham Pharmacia Biotech) and analyzed on an ALF-express automaticsequencer.

[0070] The cDNA clone contained an insert of 540 base pairs, of which344 nucleotides were sequenced.

[0071] A search for sequences homologous or identical to these 344nucleotides in a gene database from INCYTE revealed a template of 2098nucleotides, containing an open reading frame in the 3′ part of thesequence, but lacking a stop codon. This template was used to search foroverlapping sequences. The templates found in this way were assembledand hand-edited to reveal a sequence of 3835 nucleotides (SEQ ID NO:2)with an open reading frame of 473 amino acids coding for a protein witha calculated molecular weight of 53.3 kDa (SEQ ID NO:1).

Example 4

[0072] RT-PCR

[0073] To further validate the expression profile found in the INDB,RT-PCR analysis on 10 ruptured and 10 stable plaques was performed. Toexclude patient- and artery-biased expression, plaques originated fromseveral arteries of different patients (Table 1). Expression wasnormalized to the expression level of GAPDH, which expression level wascomparable between different samples (FIG. 2).

[0074] Procedure: Isolation of total RNA was carried out as describedabove. The SMART™ PCR cDNA Synthesis Kit (Clontech) was used for thepreparation of double stranded cDNA from 0.5 μg template RNA. cDNA wasdiluted to a total volume of 50 μl. PCR amplification of thepolynucleotide (sense: 5′-GGCTAATTCGGGAGATAGCC-3′, SEQ ID NO: 11,+antisense: 5′-CAACACCTCATGGCAAGTCC-3′, SEQ ID NO: 12) was performed on1 μl of first strand cDNA using standard conditions (30 cycles ofdenaturation for 1 min at 94° C., annealing for 1 min at 55° C. andextension for 1 min at 72° C.). Resulting PCR products of approximately300 bp were analyzed on a 1% agarose gel.

[0075] Result: Expression of the polynucleotide of SEQ ID NO: 2 wasfound in 8 out of 10 ruptured plaques, while only 2 out of 10 stableplaques tested positive. TABLE 1 Patient characteristics plaque type Nosex* age Artery used for Ruptured 1 m 60 Abdominal aorta RT-PCR^(†) 2 f66 Common femoral RT-PCR artery 3 m 72 Abdominal aortaSSH^(‡)/INDB^(§)/RT-PCR 4 m 74 Abdominal aorta RT-PCR 5 m 73 Abdominalaorta SSH/INDB/RT-PCR 6 m 55 Femoral artery RT-PCR 7 m 75 Abdominalaorta SSH/INDB/RT-PCR 8 m 73 Femoral artery RT-PCR 9 m 63 Abdominalaorta RT-PCR 10 m 58 Carotid artery RT-PCR Stable 11 m 72 Carotid arteryRT-PCR 12 f 67 Carotid artery RT-PCR 13 m 57 Carotid artery RT-PCR 14 f71 Femoral artery RT-PCR 15 m 78 Common femoral SSH/INDB/RT-PCR artery16 m 78 Common iliac SSH/INDB/RT-PCR artery 17 m 60 Abdominal aortaRT-PCR 18 m 68 Carotid artery RT-PCR 19 m 67 Carotid arterySSH/INDB/RT-PCR 20 m 70 Carotid artery RT-PCR

Example 5

[0076] SSH6: A Vasular Smooth Muscle Cell Specific mRNA and ProteinPlaque rupture of atherosclerotic plaques is the predominant underlyingprocess in the pathogenesis of acute coronary syndromes and peripheralvascular disease. Insight into the pathways that destabilize plaques issparse. Suppressive Subtractive Hybridization (SSH) analysis on humanatherosclerotic plaques-derived RNA, resulted in the identification of alarge library of cDNAs differentially expressed in stable and rupturedatherosclerotic plaques (see Example 1). Differential expression of thesequences of the SSH library was verified by inverse northern dot blot(INDB) or macro-array analysis (see Example 2). One of these cDNAclones, SSH 6, was over 2 fold upregulated in ruptured plaques. Thisclone contained a cDNA insert of 436 bp (the nucleotides 905-1341 of SEQID NO:2), containing a putative ORF of 57 amino acids (amino acids 1-57of SEQ ID NO:1).

[0077] To further validate the expression profile found in INDB, RT-PCRanalysis on 10 ruptured and 10 stable plaques was performed. To excludepatient- and artery-biased expression, plaques originated from severalarteries of individual patients (see Table 1, Example 4). Expression wasnormalized to the expression level of GAPDH, which was comparablebetween different samples. Expression of this sequence was found in 8out of 10 ruptured plaques, while only 2 out of 10 stable plaques testedpositive. (see Example 4)

[0078] RT-PCR on individual samples of veins (n=5), non-diseased artery(n=4) and early atherosclerotic plaques (n=5) revealed SSH6 expressionin all veins, in 50% of non-diseased arteries, and in 40% of earlylesions, respectively (FIG. 4).

[0079] A search for sequences homologous or identical to the SSH 6sequence revealed several templates, including a template of 2098nucleotides in a INCYTE gene database, showing partial overlap. However,clone SSH 6 contained an insert of 120 nt (the nucleotides 1112-1231 ofSEQ ID NO: 2) in comparison to the majority of sequences in thedatabases. This 120 nt insert contains a putative start codon (thenucleotides 1169-1171 of SEQ ID NO: 2) in frame with a large ORF. Inorder to obtain a full length SSH 6 clone, a Vascular Smooth MuscleDerived (VSMC) derived cDNA library was screened with the original cDNAfragment (kindly provided by Dr C A de Vries, AMC, Amnsterdam). Thisscreening resulted in the isolation of numerous (>10) cDNA clones, allcontaining over 2000 bp of SSH6 sequences. Sequence analysis of thelargest clone revealed the presence of a 2858 nt cDNA fragment(identical to the nucleotides 64-2920 of SEQ ID: NO 2, with theexception that in this fragment an additional “g” nucleotide is presentbetween nucleotides 2904 and 2905 of SEQ ID NO:2, which is in thenon-coding part of the sequence), containing an ORF of 473 amino acids(SEQ ID: NO 1).

[0080] Detailed bio-infornatics using public domain and INCYTE databasesindicated the presence of a putative “vascular wall specific” mRNA andvascular wall specific protein, further indicated as SSH6v. Aligmnent ofSSH6v cDNA and genomic databases showed that the SSH6 gene is spanning agenomic region of over 90 Kb on chromosome 5 p13 and consists of atleast 12 exons (see Table 2). TABLE 2 Genomic organization of SSH6Position (from nucleotide . . . to . . . in exon SEQ ID NO: 2) intronlength 1    1-158  1 1236 bp 2  159-1111 2 >37 Kb 3 1112-1231 3 >16 Kb 41232-1356 4 128 bp 5 1357-1579 5 4644 bp 6 1580-1646 6 >10 Kb 71647-1831 7 590 bp 8 1832-1972 8 >10 Kb 9 1973-2140 9 >1.5 Kb 102141-2328 10 >10 Kb 11 2329-2431 11 1311 12 2432->2920

[0081] The vascular wall specific mRNA/protein SSH6v and theubiquitously expressed SSH6 mRNA result from alternative splicing ofexon 3 (nucleotides 1112-1231 of SEQ ID NO: 2). The differential issuedistribution of SSH6v and SSH6 was further substantiated by multi-tissuenorthern blot analysis on 62 adult human tissues, 8 human cell lines, 7fetal tissues and 6 controls (Multiple Tissue Expression Array MTE:Clontech, Palo Alto, Calif., USA). FIG. 5 shows the tissue distributionof SSH 6 (using the 436 bp cDNA insert—the nucleotides 905-1341—of SEQID NO: 2 as a probe), while FIG. 6 indicates the vascular wall specificexpression of the SSH6v messenger (using a 120 nt exon-3 specificprobe). The bottom panel of FIG. 6 indicates hybridization of this probeto VSMC derived RNA and a positive control (full length SSH6v cDNA).

[0082] In order to develop immunological tools to characterize the SSH6protein in more detail, part of the ORF (909 bp) was fused toglutathione S-transferase (67 kDa) and the resulting recombinant proteinwas used to select SSH6-specific single-chain Fv fragments (scFv) (seeFIG. 7 for a schematical representation). Western blot analysis of humanatherosclerotic plaques, human plasma, human tissue lysate and severalcell lines using a SSH6 specific scFv revealed the presence of a proteinof the expected size (˜53 kDa) in vascluar wall derived lysates andplasma only, and a 35 kDa product in the majority of lysates (see FIG.8). This 35 kDa protein most likely is the result of translation startat an internal Methionine (AA 172) Furthermore, immunohistochemicalanalysis of human ruptured atherosclerotic plaques indicated SSH6protein expression in vascular smooth muscle cells (VSMC) (see FIG. 9).Interestingly, localization of the SSH6 protein nicely correlates to theobserved presence of SSH6 mRNA in primary cultures of human VSMC derivedfrom a ruptured plaque.

[0083] Conclusion: a previously unknown vascular wall specificmRNA/protein SSH6v has been identified that is expressed in human VSMCand is upregulated in ruptured atherosclerotic plaques.

Materials and Methods Example 5

[0084] RT-PCR Analysis

[0085] To reveal the expression profile of SSH6v in the vascular wall,RT-PCR was performed on mRNA isolated from veins and arteries withatherosclerotic lesions in various stages and on mRNA isolated from aprimary culture of VSMC derived from ruptured atherosclerotic lesions.RNA isolation, cDNA synthesis and RT-PCR was performed as describedpreviously. In brief, a SSH6v-specific DNA fragment of 217 bp wasamplified by PCR on first strand cDNA using the sense primer(5′-GGCTAATTCGGGAGATAGCC-3′, SEQ ID NO: 11) and antisense primer(5′-CAACACCTCATGGCAAGTCC-3′, SEQ ID NO: 12) under standard conditions(30×(94° C., 1 min; 55° C., 1 min; 72° C., 1 min). The resulting PCRproducts were analyzed on a 1% agarose gel.

[0086] Multi-tissue Northern Blot Analysis

[0087] Multi-tissue northern blot was performed using the MultipleTissue Expression Array MTE (Clontech, Palo Alto, Calif., USA)essentially according the protocol of the manufacturer. Briefly, the MTEarray was hybridized with denatured ³³P-labeled cDNA probes for 12 hoursat 65° C. and exposed to x-ray film at −70° C. during 12 hours.

[0088] Construction of the Glutathione S-Transferase-SSH6 Fusion Protein(GST-SSH6) Expression Plasmid

[0089] The C-terminal part of the SSH-6 cDNA was amplified using thesense primer 5′-CCTAAATCTAGAGCGTCGACGATGCTGG-3′ (SEQ ID NO: 13) andantisense primer 5′-AAGCTGTTAGTCGACCCTTCACA-3′ (SEQ ID NO: 14) in orderto introduce a SalI restriction site for the construction of theexpression plasmid. Simultaneously with the introduction of the desiredrestriction sites, a proline (CCA) and arginine (AGG) codon inside theopen reading frame of SSH6 were mutated into a serine (TCG) andthreonine (ACG) codon, respectively. Subsequently, the PCR product wasdigested with Sal I and the resulting 938 bp fragment was ligated inpGEX-4T-2 and transformed to BL21 E. coli cells. In order to produce GSTprotein BL21 E. coli cells were transformed with pGEX4T-2 withoutadditional insert.

[0090] Western Blot Analysis of SSH6

[0091] Lysates of various human tissues and cell lines were prepared asfollows: 2-5×10⁷ cells were collected, resuspended in 500 μl ice coldlysis buffer (25 mM Tris-HCl (pH 7.5), containing 150 mM NaCl, 1 mMEDTA, 2 mM PMSF, 1 mM DTT, 0.1 mM benzamidine and 1% Nonidet P40) andincubated for 20 min on ice. The cell lysates were cleared bycentrifugation. Lysates equivalent to 10-20 μg of total proteins orserial dilutions of human plasma were separated by SDS-PAGE (9%) andtransferred onto nitrocellulose. After blocking with PBS containing 2%(w/v) skimmed milk powder (MPBS), blots were stained for 1 h with 5μg/ml of purified anti SSH6-scFv. Following incubation with HRP labeledanti-myc antibody HRP activity was visualized by ECL staining.

[0092] Immunohistochemical Analysis

[0093] Four μm frozen atherosclerotic plaques sections were pre-treatedwith TBS-TS (TBS, 0.1% (v/v) Tween 20, 3% human serum and 3% sheepserum). Subsequently, sections were incubated for 30 min with scFv-2A4.Bound scFv antibodies were detected with an anti myc-antibody (9E10),followed by incubation with biotinylated sheep anti-mouse antibody andan alkaline phosphatase coupled ABC reagent. Alkaline phosphataseactivity was visualized using the Alkaline Phosphatase Kit I (Vector)containing 1 mM levamisole (Sigma), resulting in a red precipitate. Thesections were counterstained with hematoxylin.

Example 6

[0094] According to the procedures described in Examples 1-4, also SEQID NO:4 was identified, another clone upregulated (3-fold) in unstableplaques.

[0095] Specific sequencing:

[0096] The cDNA clone contained an insert of 1050 base pairs, of which391 nucleotides were sequenced.

[0097] A search for sequences homologous or identical to these 391nucleotides in the INCYTE gene database revealed a sequence of 3145nucleotides (SEQ ID NO:4), containing an open reading frame of 946 aminoacids (SEQ ID NO:3). This open reading frame corresponds to a proteinwith similarity to the human sorting nexin (GenBank accession number AK000362).

Example 7

[0098] According to the procedures described in Examples 1-4, also SEQID NO:6 was identified, a clone downregulated in unstable plaques(specific for stable plaques). In FIG. 3, a INDB analysis of thepolynucleotide of SEQ ID NO:6 is shown.

[0099] Specific sequencing:

[0100] The cDNA clone contained an insert of 400 base pairs, of which348 nucleotides were sequenced.

[0101] A search for sequences homologous or identical to these 348nucleotides in the Geneseq patent database revealed a sequence of 4117nucleotides (SEQ ID NO:6), containing an open reading frame of 950 aminoacids (SEQ ID NO:5). This open reading frame has been predicted toencode a membrane spanning protein, MSP-5 (WO 9946380).

1 14 1 473 PRT Homo sapiens 1 Met Ala Gln His Asp Phe Ala Pro Ala TrpLeu Asn Phe Pro Thr Pro 1 5 10 15 Pro Ser Ser Thr Lys Ser Ser Leu AsnPhe Glu Lys His Ser Glu Asn 20 25 30 Phe Ala Trp Thr Glu Asn Arg Tyr AspVal Asn Arg Arg Arg His Asn 35 40 45 Ser Ser Asp Gly Phe Asp Ser Ala IleGly Arg Pro Asn Gly Gly Asn 50 55 60 Phe Gly Arg Lys Glu Lys Asn Gly TrpArg Thr His Gly Arg Asn Gly 65 70 75 80 Thr Glu Asn Ile Asn His Arg GlyGly Tyr His Gly Gly Ser Ser Arg 85 90 95 Ser Arg Ser Ser Ile Phe His AlaGly Lys Ser Gln Gly Leu His Glu 100 105 110 Asn Asn Ile Pro Asp Asn GluThr Gly Arg Lys Glu Asp Lys Arg Glu 115 120 125 Arg Lys Gln Phe Glu AlaGlu Asp Phe Pro Ser Leu Asn Pro Glu Tyr 130 135 140 Glu Arg Glu Pro AsnHis Asn Lys Ser Leu Ala Ala Gly Val Trp Glu 145 150 155 160 Tyr Pro ProAsn Pro Lys Ser Arg Ala Pro Arg Met Leu Val Ile Lys 165 170 175 Lys GlyAsn Thr Lys Asp Leu Gln Leu Ser Gly Phe Pro Val Val Gly 180 185 190 AsnLeu Pro Ser Gln Pro Val Lys Asn Gly Thr Gly Pro Ser Val Tyr 195 200 205Lys Gly Leu Val Pro Lys Pro Ala Ala Pro Pro Thr Lys Pro Thr Gln 210 215220 Trp Lys Ser Gln Thr Lys Glu Asn Lys Val Gly Thr Ser Phe Pro His 225230 235 240 Glu Ser Thr Phe Gly Val Gly Asn Phe Asn Ala Phe Lys Ser ThrAla 245 250 255 Lys Asn Phe Ser Pro Ser Thr Asn Ser Val Lys Glu Cys AsnArg Ser 260 265 270 Asn Ser Ser Ser Pro Val Asp Lys Leu Asn Gln Gln ProArg Leu Thr 275 280 285 Lys Leu Thr Arg Met Arg Thr Asp Lys Lys Ser GluPhe Leu Lys Ala 290 295 300 Leu Lys Arg Asp Arg Val Glu Glu Glu His GluAsp Glu Ser Arg Ala 305 310 315 320 Gly Ser Glu Lys Asp Asp Asp Ser PheAsn Leu His Asn Ser Asn Ser 325 330 335 Thr His Gln Glu Arg Asp Ile AsnArg Asn Phe Asp Glu Asn Glu Ile 340 345 350 Pro Gln Glu Asn Gly Asn AlaSer Val Ile Ser Gln Gln Ile Ile Arg 355 360 365 Ser Ser Thr Phe Pro GlnThr Asp Val Leu Ser Ser Ser Leu Glu Ala 370 375 380 Glu His Arg Leu LeuLys Glu Met Gly Trp Gln Glu Asp Ser Glu Asn 385 390 395 400 Asp Glu ThrCys Ala Pro Leu Thr Glu Asp Glu Met Arg Glu Phe Gln 405 410 415 Val IleSer Glu Gln Leu Gln Lys Asn Gly Leu Arg Lys Asn Gly Ile 420 425 430 LeuLys Asn Gly Leu Ile Cys Asp Phe Lys Phe Gly Pro Trp Lys Asn 435 440 445Ser Thr Phe Lys Pro Thr Thr Glu Asn Asp Asp Thr Glu Thr Ser Ser 450 455460 Ser Asp Thr Ser Asp Asp Asp Asp Val 465 470 2 3835 DNA Homo sapiens2 agaacattgc ggatcgggtc ggcgccattt tgggactgag actggttgtg ggggagggaa 60aagcggcaaa aggggattat tcaaagtacc gaaaaccttc tcccgggatc aggcgcggcg 120gcacccccag gccaggggca cctctggtgg ggcagaaggt gattgaatta ctcagatatg 180aagatcatca tctaggtttt gtgtaaaagg ccctggatat tttaagtggc cattttggat 240ttacagtgtt tttggataat tttgccccag aagtttatta aaattggcaa gaatcgtctg 300tgaagtgaat tgatagtagt gaacaattca gcaagctact taaaaagaga cccaggcagc 360atttcttcag tattttggtt caaacggatt atataactgg ttacagtatt tcagctggtg 420gtaatttttg cctccccttc ccccaccccg ttgttggggt tcttcagccg aaactgagag 480acgttgattt gtgtactgag tagtttcagc agtttcaaat gactgagtat tgctgaagtt 540tcatggcagt ttatttttac ctttattgaa agttttagga atttttgact tcagctcttt 600catgtcacaa tgggacactt tttctgaatg aagagattga aagaatacag agtttttttc 660cttttatctt ttatttacgt ggaaatttaa gatgttgcag ttttccggca gcatggtagt 720attgagatag ctatgtgtgt ctctgtatat gctgatgttt aggaatgctc ttcagatgtg 780aaattttctt tttgtttttg ctttttggct cgtaaattgg atatttcatc tggagtggac 840aagtacaaca gtggcaagta catggaataa taaagaagac tttgatctta aatctaaaga 900acttggctaa ttcgggagat agccatatga aaactttaaa acagaagtat gggtagctga 960cttgaagtaa ctctatgtca aatagtcgta ggttaagtat cttcaaagaa cttcgatatt 1020atttcagagg atacaaaata aaaatacaaa ctggaaaata aagattacag agaaaaaacc 1080aacaccttcc tgtgcagtcc tgttggaatt tggacttgcc atgaggtgtt gaagccttgt 1140ttcactgagt tggagagact ggacctaaat ggcgcagcat gactttgctc cagcctggct 1200taatttccct actccaccat catcaacaaa gtcgtcattg aattttgaga agcattctga 1260aaactttgca tggacagaga atcgttatga tgtgaaccgt cgacgacaca actcttcaga 1320tggctttgat tctgctattg gacgtcctaa tggaggtaac tttggaagga aagaaaaaaa 1380tggatggcgt acacatggaa gaaatggtac agaaaacata aatcatcgag gtggatacca 1440tggtggaagt tcccgttctc gtagcagtat tttccatgca ggaaaaagcc aaggactaca 1500tgaaaacaac atacctgaca atgaaaccgg gaggaaagaa gacaagagag aacgcaaaca 1560gtttgaagct gaggattttc cgtctttaaa tcctgagtat gagagagaac caaatcacaa 1620taagtcttta gctgcaggtg tgtgggaata tcctccgaat cctaaatcta gagctccaag 1680gatgctggtc attaagaaag gtaatacaaa agacttacag ctatctggat tcccagtagt 1740aggaaatctt ccgtcacagc cagttaagaa tggaactggt ccaagtgttt ataaaggttt 1800agtccctaaa cctgctgctc cacctacaaa acctacacaa tggaaaagcc aaacaaaaga 1860aaataaagtt ggaacttctt tccctcatga gtccacattt ggcgttggca actttaatgc 1920ttttaaatca actgccaaga actttagtcc atctacaaat tcagtgaaag agtgtaatcg 1980ctcaaattcc tcttctcctg ttgacaaact taatcagcag cctcgtctaa ccaaactgac 2040acgaatgcgc actgataaga agagtgaatt tttgaaagca ttgaaaagag acagagtaga 2100agaggaacat gaagatgaaa gccgtgctgg ctcagagaag gatgacgact catttaattt 2160acataacagc aatagtactc accaagaaag ggatataaac cgaaacttcg atgaaaatga 2220aattcctcaa gagaatggca atgcctcagt gatttcccag cagatcattc ggtcttcaac 2280cttcccacaa actgatgttc tttcaagttc acttgaggca gaacacagat tgttaaagga 2340aatgggctgg caggaagaca gtgaaaatga tgaaacatgt gctcccttaa ctgaggatga 2400aatgagagaa ttccaagtta ttagtgaaca gttacagaag aatggtctga gaaaaaatgg 2460tattttgaaa aatggcttga tctgtgactt caagtttgga ccgtggaaga acagcacttt 2520caaacccaca actgagaatg atgacacaga gacaagtagc agtgatacat cagatgacga 2580cgatgtgtga aggatttcct aacagcttta gaaatcttag tgtgatacat ctctcataca 2640gtttggggtg aattgtaaaa atgaagaact ataatttatg tagtgaaata ccccattaga 2700agaggatttt ttgggggact tcaatatgaa gaaaaccaag aatgttttgt tgggctgtgt 2760tgaacattat ttctttgtaa atgaatgttg taggaatgag gacttgggtt ggtccaacat 2820tgactttctt catcactgca acatttctct gactagcaat gtgacgatgt aacaaatgag 2880attttctcat ttaataataa aaaattgtgt aatgttttgc aaagcttctg tcttaaaatg 2940tccaggtctt aagaaaaaag gcagcttaca ctgttttgct tgcagagtca tatctttttc 3000gtacaatgga aatcctcaag tccactttgt gcggtctccc tctccttccc ccaaaaaaca 3060acaacaacaa aacaaaaacc aaaaaggaaa atgtagcatg ttggctaaaa ctggagcaaa 3120gtgcactaaa acaatttcct gaactcacct gttgtactat tcacctttta aaccataaat 3180tgctctttag ccatttgtag tgcagtaaat gttacaggaa aagacttggc acattttctt 3240ccaaatttta agaggtgatt ttcaaaagct ttattggggt atgttgtcag accagggttt 3300tcagagttga tggaaaagag tcttgtgaga aaacttattt tgataaatta ttacacacgc 3360agaaaaactg atcacactga ctggatctgt ccacgacatg gaaaataaac tggattttca 3420gaatattgtt gttttctgta gtgttcaagg tattgtttct aaacataaac atactctaaa 3480catgctttat tcacttgtta aagtcatact tttaaaagta ataccttact aaagatggtg 3540attacttttc cgaggtcaga aaaggaaagc taagcgtttt cattatcaaa tacacaagct 3600tattaaatga atgactgtta actactttat tttcatttgc acattaattt tggaattgtt 3660tctgttttgc tgctgacgga aatactattt tggctctgtg tatatttgta ttttgatttt 3720tctggtttgt ttacccccat ttgcttttag ctccccctta tgtttaaata tattctaact 3780tatgtaaaga gcataatctt agagcaaaaa tacttgaggt tttatgtcag atcta 3835 3 946PRT Homo sapiens 3 Met Val Pro Trp Val Arg Thr Met Gly Gln Lys Leu LysGln Arg Leu 1 5 10 15 Arg Leu Asp Val Gly Arg Glu Ile Cys Arg Gln TyrPro Leu Phe Cys 20 25 30 Phe Leu Leu Leu Cys Leu Ser Ala Ala Ser Leu LeuLeu Asn Arg Tyr 35 40 45 Ile His Ile Leu Met Ile Phe Trp Ser Phe Val AlaGly Val Val Thr 50 55 60 Phe Tyr Cys Ser Leu Gly Pro Asp Ser Leu Leu ProAsn Ile Phe Phe 65 70 75 80 Thr Ile Lys Tyr Lys Pro Lys Gln Leu Gly LeuGln Glu Leu Phe Pro 85 90 95 Gln Gly His Ser Cys Ala Val Cys Gly Lys ValLys Cys Lys Arg His 100 105 110 Arg Pro Ser Leu Leu Leu Glu Asn Tyr GlnPro Trp Leu Asp Leu Lys 115 120 125 Ile Ser Ser Lys Val Asp Ala Ser LeuSer Glu Val Leu Glu Leu Val 130 135 140 Leu Glu Asn Phe Val Tyr Pro TrpTyr Arg Asp Val Thr Asp Asp Glu 145 150 155 160 Ser Phe Val Asp Glu LeuArg Ile Thr Leu Arg Phe Phe Ala Ser Val 165 170 175 Leu Ile Arg Arg IleHis Lys Val Asp Ile Pro Ser Ile Ile Thr Lys 180 185 190 Lys Leu Leu LysAla Ala Met Lys His Ile Glu Val Ile Val Lys Ala 195 200 205 Arg Gln LysVal Lys Asn Thr Glu Phe Leu Gln Gln Ala Ala Leu Glu 210 215 220 Glu TyrGly Pro Glu Leu His Val Ala Leu Arg Ser Arg Arg Asp Glu 225 230 235 240Leu His Tyr Leu Arg Lys Leu Thr Glu Leu Leu Phe Pro Tyr Ile Leu 245 250255 Pro Pro Lys Ala Thr Asp Arg Arg Ser Leu Thr Leu Leu Ile Arg Glu 260265 270 Ile Leu Ser Gly Ser Val Phe Leu Pro Ser Leu Asp Phe Leu Ala Asp275 280 285 Pro Asp Thr Val Asn His Leu Leu Ile Ile Phe Ile Asp Asp SerPro 290 295 300 Pro Glu Lys Ala Thr Glu Pro Ala Ser Pro Leu Val Pro PheLeu Gln 305 310 315 320 Lys Phe Ala Glu Pro Arg Asn Lys Lys Pro Ser ValLeu Lys Leu Glu 325 330 335 Leu Lys Gln Ile Arg Glu Gln Gln Asp Leu LeuPhe Arg Phe Met Asn 340 345 350 Phe Leu Lys Gln Glu Gly Ala Val His ValLeu Gln Phe Cys Leu Thr 355 360 365 Val Glu Glu Phe Asn Asp Arg Ile LeuArg Pro Glu Leu Ser Asn Asp 370 375 380 Glu Met Leu Ser Leu His Glu GluLeu Gln Lys Ile Tyr Lys Thr Tyr 385 390 395 400 Cys Leu Asp Glu Ser IleAsp Lys Ile Arg Phe Asp Pro Phe Ile Val 405 410 415 Glu Glu Ile Gln ArgIle Ala Glu Gly Pro His Ile Asp Val Val Lys 420 425 430 Leu Gln Thr MetArg Cys Leu Phe Glu Ala Tyr Glu His Val Leu Ser 435 440 445 Leu Leu GluAsn Val Phe Thr Pro Met Phe Cys His Ser Asp Glu Tyr 450 455 460 Phe ArgGln Leu Leu Arg Gly Ala Glu Ser Pro Thr Arg Asn Ser Lys 465 470 475 480Leu Asn Arg Gly Ser Leu Ser Leu Asp Asp Phe Arg Asn Thr Gln Lys 485 490495 Arg Gly Glu Ser Phe Gly Ile Ser Arg Ile Gly Ser Lys Ile Lys Gly 500505 510 Val Phe Arg Ser Thr Thr Met Glu Gly Ala Met Leu Pro Asn Tyr Gly515 520 525 Val Ala Glu Gly Glu Asp Asp Phe Ile Glu Glu Gly Ile Val ValMet 530 535 540 Glu Asp Asp Ser Pro Val Glu Ala Val Ser Thr Pro Asn ThrPro Arg 545 550 555 560 Asn Leu Ala Ala Trp Lys Ile Ser Ile Pro Tyr ValAsp Phe Phe Glu 565 570 575 Asp Pro Ser Ser Glu Arg Lys Glu Lys Lys GluArg Ile Pro Val Phe 580 585 590 Cys Ile Asp Val Glu Arg Asn Asp Arg ArgAla Val Gly His Glu Pro 595 600 605 Glu His Trp Ser Val Tyr Arg Arg TyrLeu Glu Phe Tyr Val Leu Glu 610 615 620 Ser Lys Leu Thr Glu Phe His GlyAla Phe Pro Asp Ala Gln Leu Pro 625 630 635 640 Ser Lys Arg Ile Ile GlyPro Lys Asn Tyr Glu Phe Leu Lys Ser Lys 645 650 655 Arg Glu Glu Phe GlnGlu Tyr Leu Gln Lys Leu Leu Gln His Pro Glu 660 665 670 Leu Ser Asn SerGln Leu Leu Ala Asp Phe Leu Ser Pro Asn Gly Gly 675 680 685 Glu Thr GlnPhe Leu Asp Lys Ile Leu Pro Asp Val Asn Leu Gly Lys 690 695 700 Ile IleLys Ser Val Pro Gly Lys Leu Met Lys Glu Lys Gly Gln His 705 710 715 720Leu Glu Pro Phe Ile Met Asn Phe Ile Asn Ser Cys Glu Ser Pro Lys 725 730735 Pro Lys Pro Ser Arg Pro Glu Leu Thr Ile Leu Ser Pro Thr Ser Glu 740745 750 Asn Asn Lys Lys Leu Phe Asn Asp Leu Phe Lys Asn Asn Ala Asn Arg755 760 765 Ala Glu Asn Thr Glu Arg Lys Gln Asn Gln Asn Tyr Phe Met GluVal 770 775 780 Met Thr Val Glu Gly Val Tyr Asp Tyr Leu Met Tyr Val GlyArg Val 785 790 795 800 Val Phe Gln Ile Pro Asp Trp Leu His His Leu LeuMet Gly Thr Arg 805 810 815 Ile Leu Phe Lys Asn Thr Leu Glu Met Tyr ThrAsp Tyr Tyr Leu Gln 820 825 830 Cys Lys Leu Glu Gln Leu Phe Gln Glu HisArg Leu Val Ser Leu Ile 835 840 845 Thr Leu Leu Arg Asp Ala Ile Phe CysGlu Asn Thr Glu Pro Arg Ser 850 855 860 Leu Gln Asp Lys Gln Lys Gly AlaLys Gln Thr Phe Glu Glu Met Met 865 870 875 880 Asn Tyr Ile Pro Asp LeuLeu Val Lys Cys Ile Gly Glu Glu Thr Lys 885 890 895 Tyr Glu Ser Ile ArgLeu Leu Phe Asp Gly Leu Gln Gln Pro Val Leu 900 905 910 Asn Lys Gln LeuThr Tyr Val Leu Leu Asp Ile Val Ile Gln Glu Leu 915 920 925 Phe Pro GluLeu Asn Lys Val Gln Lys Glu Val Thr Ser Val Thr Ser 930 935 940 Trp Met945 4 3145 DNA Homo sapiens 4 aaaaaactgc cggtaagcgt ctgtgtgcgccgccaagtcg gtggggcggg gacgcgaggt 60 gtggatgggg ggtcgccttg acctctgcctcagccagtag cgcagcctcg gcctcgccgt 120 tacggagatg gtgccctggg tgcggacgatggggcagaag ctgaagcagc ggctgcgact 180 ggacgtggga cgcgagatct gccgccagtacccgctgttc tgcttcctgc tgctctgtct 240 cagcgccgcc tccctgcttc ttaacaggtatattcatatt ttaatgatct tctggtcatt 300 tgttgctgga gttgtcacat tctactgctcactaggacct gattctctct taccaaatat 360 attcttcaca ataaaataca aacccaagcagttaggactt caggaattat ttcctcaagg 420 tcatagctgt gctgtttgtg gtaaagtgaaatgtaaacga cataggcctt ctttgctact 480 tgaaaactac cagccatggc tagacctgaaaatttcttcc aaggttgatg catctctctc 540 agaggttctt gaattagtgt tggaaaactttgtttatccg tggtacaggg atgtgacaga 600 tgatgaatcc tttgttgatg aactgagaataacattacgt ttttttgcat ctgtcttaat 660 aagaaggatt cacaaggtgg atattccatctattataacc aagaaactat taaaagcagc 720 aatgaagcat atagaagtga tagttaaagccagacagaaa gtaaaaaata cagagttttt 780 acagcaagct gctttagaag aatatggtccagagcttcat gttgctttga gaagtcgaag 840 agatgaattg cactatttaa ggaaacttactgaactgctt tttccttata ttttgcctcc 900 taaagcaaca gaccgcagat ctctgaccttacttataaga gagattctgt ctggctctgt 960 gttccttcct tctttggatt tcctagctgatccagatact gtgaatcatt tgcttatcat 1020 cttcatagat gacagtccac ctgaaaaagcaactgaaccg gcttctcctt tggttccatt 1080 cttgcagaaa tttgcagaac ctagaaataaaaagccatct gtgctgaagt tagaattgaa 1140 gcaaatcaga gagcaacaag atcttttatttcgttttatg aactttctga aacaagaagg 1200 cgcagtgcac gtgttgcagt tttgtttgactgtggaggaa tttaatgata gaattttacg 1260 accagaatta tcaaatgatg aaatgctgtctcttcatgaa gaattgcaga agatttataa 1320 aacatactgt ttggatgaaa gtattgacaaaattagattt gatcccttca ttgtagaaga 1380 gattcaaaga attgctgaag gcccacacatagatgttgtg aaacttcaaa ctatgagatg 1440 tctttttgaa gcatatgaac atgttctttcccttttggag aatgtattta ctcctatgtt 1500 ctgccatagt gatgagtatt tcagacaacttttaagaggt gcagaatcac caacacgcaa 1560 ttcaaaattg aacagaggta gcctaagtttggatgatttt cggaacacac agaaaagggg 1620 agaatcattt ggaatcagca gaataggtagcaaaattaaa ggagtattca gaagtaccac 1680 aatggaggga gctatgttgc ctaattatggtgtagctgaa ggtgaagatg attttattga 1740 agaaggtatt gttgtaatgg aagatgattctccagtggag gctgtgagca cacctaatac 1800 tccccgaaac cttgctgcat ggaaaattagcattccatat gtagactttt ttgaggatcc 1860 ctcctctgaa aggaaggaga aaaaagaaagaattcctgtg ttttgtattg atgttgaaag 1920 aaatgataga agagcagttg gacacgagcctgaacattgg tctgtctata gaagatatct 1980 tgaattctat gtacttgaat caaaactaacagaatttcat ggtgcatttc ctgatgccca 2040 gcttccttct aagaggatca ttggccccaaaaattatgaa ttcttaaagt caaagaggga 2100 agagttccaa gaatatctac agaaacttctgcagcatcca gaactgagta atagtcaact 2160 tctggcagac tttctttccc ctaatggtggggaaacacaa tttcttgata agatactacc 2220 agatgtaaat cttgggaaaa ttataaaatctgttcctgga aaactaatga aagagaaagg 2280 tcagcatttg gaacctttta tcatgaatttcattaattct tgtgagtctc caaagcctaa 2340 accaagtaga ccagaactga ccattctcagccctacttca gaaaacaaca agaagctttt 2400 caatgatctg tttaaaaata atgcaaaccgtgctgaaaat acagagagaa agcaaaatca 2460 gaattatttt atggaggtga tgactgtagaaggagtctat gattacctga tgtatgtagg 2520 acgggtagtt ttccagattc ctgactggcttcatcatctc ttaatgggaa ctcgaatcct 2580 ctttaaaaac accctggaaa tgtatactgattactatctt cagtgtaaac tagaacagct 2640 atttcaggag caccgtttgg tctcactcataacacttctc agagatgcta tattctgtga 2700 aaacactgaa cctcgctctc tccaagataagcaaaaagga gcaaaacaga cttttgaaga 2760 aatgatgaat tacattccag atctgttagtcaagtgtatt ggtgaggaaa ccaagtatga 2820 aagcatcaga cttctgtttg atggcttacagcaaccagta ctcaacaagc agctgactta 2880 tgttttattg gacattgtga tacaggaactgtttccagag ctcaataagg tacaaaagga 2940 agttacctct gtgacatctt ggatgtaaacacttggattt ggtatagaat aacccattga 3000 aatttctgct gtgcgagggt ggtagaaatttacttttttg ggtatattct atatatatta 3060 tgtacatcgc tgtctgaaat tttagttattttttgttttt aataaagact aacacaactt 3120 aatgattaaa aaaaaaaaaa aaaaa 3145 5950 PRT Homo sapiens 5 Met Thr Trp Arg Met Gly Pro Arg Phe Thr Met LeuLeu Ala Met Trp 1 5 10 15 Leu Val Cys Gly Ser Glu Pro His Pro His AlaThr Ile Arg Gly Ser 20 25 30 His Gly Gly Arg Lys Val Pro Leu Val Ser ProAsp Ser Ser Arg Pro 35 40 45 Ala Arg Phe Leu Arg His Thr Gly Arg Ser ArgGly Ile Glu Arg Ser 50 55 60 Thr Leu Glu Glu Pro Asn Leu Gln Pro Leu GlnArg Arg Arg Ser Val 65 70 75 80 Pro Val Leu Arg Leu Ala Arg Pro Thr GluPro Pro Ala Arg Ser Asp 85 90 95 Ile Asn Gly Ala Ala Val Arg Pro Glu GlnArg Pro Ala Ala Arg Gly 100 105 110 Ser Pro Arg Glu Met Ile Arg Asp GluGly Ser Ser Ala Arg Ser Arg 115 120 125 Met Leu Arg Phe Pro Ser Gly SerSer Ser Pro Asn Ile Leu Ala Ser 130 135 140 Phe Ala Gly Lys Asn Arg ValTrp Val Ile Ser Ala Pro His Ala Ser 145 150 155 160 Glu Gly Tyr Tyr ArgLeu Met Met Ser Leu Leu Lys Asp Asp Val Tyr 165 170 175 Cys Glu Leu AlaGlu Arg His Ile Gln Gln Ile Val Leu Phe His Gln 180 185 190 Ala Gly GluGlu Gly Gly Lys Val Arg Arg Ile Thr Ser Glu Gly Gln 195 200 205 Ile LeuGlu Gln Pro Leu Asp Pro Ser Leu Ile Pro Lys Leu Met Ser 210 215 220 PheLeu Lys Leu Glu Lys Gly Lys Phe Gly Met Val Leu Leu Lys Lys 225 230 235240 Thr Leu Gln Val Glu Glu Arg Tyr Pro Tyr Pro Val Arg Leu Glu Ala 245250 255 Met Tyr Glu Val Ile Asp Gln Gly Pro Ile Arg Arg Ile Glu Lys Ile260 265 270 Arg Gln Lys Gly Phe Val Gln Lys Cys Lys Ala Ser Gly Val GluGly 275 280 285 Gln Val Val Ala Glu Gly Asn Asp Gly Gly Gly Gly Ala GlyArg Pro 290 295 300 Ser Leu Gly Ser Glu Lys Lys Lys Glu Asp Pro Arg ArgAla Gln Val 305 310 315 320 Pro Pro Thr Arg Glu Ser Arg Val Lys Val LeuArg Lys Leu Ala Ala 325 330 335 Thr Ala Pro Ala Leu Pro Gln Pro Pro SerThr Pro Arg Ala Thr Thr 340 345 350 Leu Pro Pro Ala Pro Ala Thr Thr ValThr Arg Ser Thr Ser Arg Ala 355 360 365 Val Thr Val Ala Ala Arg Pro MetThr Thr Thr Ala Phe Pro Thr Thr 370 375 380 Gln Arg Pro Trp Thr Pro SerPro Ser His Arg Pro Pro Thr Thr Thr 385 390 395 400 Glu Val Ile Thr AlaArg Arg Pro Ser Val Ser Glu Asn Leu Tyr Pro 405 410 415 Pro Ser Arg LysAsp Gln His Arg Glu Arg Pro Gln Thr Thr Arg Arg 420 425 430 Pro Ser LysAla Thr Ser Leu Glu Ser Phe Thr Asn Ala Pro Pro Thr 435 440 445 Thr IleSer Glu Pro Ser Thr Arg Ala Ala Gly Pro Gly Arg Phe Arg 450 455 460 AspAsn Arg Met Asp Arg Arg Glu His Gly His Arg Asp Pro Asn Val 465 470 475480 Val Pro Gly Pro Pro Lys Pro Ala Lys Glu Lys Pro Pro Lys Lys Lys 485490 495 Ala Gln Asp Lys Ile Leu Ser Asn Glu Tyr Glu Glu Lys Tyr Asp Leu500 505 510 Ser Arg Pro Thr Ala Ser Gln Leu Glu Asp Glu Leu Gln Val GlyAsn 515 520 525 Val Pro Leu Lys Lys Ala Lys Glu Ser Lys Lys His Glu LysLeu Glu 530 535 540 Lys Pro Glu Lys Glu Lys Lys Lys Lys Met Lys Asn GluAsn Ala Asp 545 550 555 560 Lys Leu Leu Lys Ser Glu Lys Gln Met Lys LysSer Glu Lys Lys Ser 565 570 575 Lys Gln Glu Lys Glu Lys Ser Lys Lys LysLys Gly Gly Lys Thr Glu 580 585 590 Gln Asp Gly Tyr Gln Lys Pro Thr AsnLys His Phe Thr Gln Ser Pro 595 600 605 Lys Lys Ser Val Ala Asp Leu LeuGly Ser Phe Glu Gly Lys Arg Arg 610 615 620 Leu Leu Leu Ile Thr Ala ProLys Ala Glu Asn Asn Met Tyr Val Gln 625 630 635 640 Gln Arg Asp Glu TyrLeu Glu Ser Phe Cys Lys Met Ala Thr Arg Lys 645 650 655 Ile Ser Val IleThr Ile Phe Gly Pro Val Asn Asn Ser Thr Met Lys 660 665 670 Ile Asp HisPhe Gln Leu Asp Asn Glu Lys Pro Met Arg Val Val Asp 675 680 685 Asp GluAsp Leu Val Asp Gln Arg Leu Ile Ser Glu Leu Arg Lys Glu 690 695 700 TyrGly Met Thr Tyr Asn Asp Phe Phe Met Val Leu Thr Asp Val Asp 705 710 715720 Leu Arg Val Lys Gln Tyr Tyr Glu Val Pro Ile Thr Met Lys Ser Val 725730 735 Phe Asp Leu Ile Asp Thr Phe Gln Ser Arg Ile Lys Asp Met Glu Lys740 745 750 Gln Lys Lys Glu Gly Ile Val Cys Lys Glu Asp Lys Lys Gln SerLeu 755 760 765 Glu Asn Phe Leu Ser Arg Phe Arg Trp Arg Arg Arg Leu LeuVal Ile 770 775 780 Ser Ala Pro Asn Asp Glu Asp Trp Ala Tyr Ser Gln GlnLeu Ser Ala 785 790 795 800 Leu Ser Gly Gln Ala Cys Asn Phe Gly Leu ArgHis Ile Thr Ile Leu 805 810 815 Lys Leu Leu Gly Val Gly Glu Glu Val GlyGly Val Leu Glu Leu Phe 820 825 830 Pro Ile Asn Gly Ser Ser Val Val GluArg Glu Asp Val Pro Ala His 835 840 845 Leu Val Lys Asp Ile Arg Asn TyrPhe Gln Val Ser Pro Glu Tyr Phe 850 855 860 Ser Met Leu Leu Val Gly LysAsp Gly Asn Val Lys Ser Trp Tyr Pro 865 870 875 880 Ser Pro Met Trp SerMet Val Ile Val Tyr Asp Leu Ile Asp Ser Met 885 890 895 Gln Leu Arg ArgGln Glu Met Ala Ile Gln Gln Ser Leu Gly Met Arg 900 905 910 Cys Pro GluAsp Glu Tyr Ala Gly Tyr Gly Tyr His Ser Tyr His Gln 915 920 925 Gly TyrGln Asp Gly Tyr Gln Asp Asp Tyr Arg His His Glu Ser Tyr 930 935 940 HisHis Gly Tyr Pro Tyr 945 950 6 4117 DNA Homo sapiens 6 taagttaccccgatgacttg gtttggaagg ggttaaggca ccagtcatcc tcttctaaag 60 tgatttatgatgatgtgtgg agtttaaaaa ctttacccca ccccaaagaa cagccctctc 120 actcctcactgagtccactc tgaacgtgct aaaatgggaa ggaggcggtg ttttgatgat 180 ctgttaaattcttagtgaag tttccttgat ttccagtggc tgctgttgtt tgagtttggt 240 ttggagcaaaactgaggtag tcctaacatt tctgggactg aatccaggca agagaaagaa 300 gaaaaagaagaagaaaaaga ggaggaaaaa ggtagggaga aataaaggga ggagagaagc 360 acagtgaaaaaaaaaaaaag tcccttttcg acatcacatt cctgtgtttt ccctcagcct 420 ggaaaacatatgaatcccag tgcttttacg cccggaaaca aagagactaa gccagactat 480 gggggaaagggagataagaa ggatcctgga actttaaaga gggaaagagt gagattcaga 540 aatcgccaggactggacttt aagggacgtc ctgtgtcagc acaagggact ggcacacaca 600 gacacacgagaccgaggaga aactgcagac aaatggagat acaaagactt agaaggacag 660 ctcctttcacctcatcctac ttgtccagaa ggtaaaaaga cacagccaga aagaaaaggc 720 atcggctcagctctcagatc aggacaggct gtggatctgt ggcggtactc tgaaagctgg 780 agctgcagcacacccctttt gtattgctca ccctcggtaa agagagagag ggctgggagg 840 aaaagtagttcatctaggaa actgtcctgg gaaccaaact tctgatttct tttgcaaccc 900 tctgcattccatctctatga gccaccattg gattacacaa tgacatggag aatgggaccc 960 cgtttcactatgctgttggc catgtggcta gtgtgtggat cagaacccca cccccatgcc 1020 actattagaggcagccacgg aggacggaaa gtgcctttgg tttctccgga cagcagtagg 1080 ccagctcggtttctgaggca cactgggagg tctcgcggaa ttgagagatc cactctggag 1140 gaaccaaaccttcagcctct ccagagaagg aggagtgtgc ccgtgttgag actagctcgc 1200 ccaacagagccgccagcccg ctcggacatc aatggggccg ccgtgagacc tgagcaaaga 1260 ccagcagccaggggctctcc gcgtgagatg atcagagatg aggggtcctc agctcggtca 1320 agaatgttgcgtttcccttc ggggtccagc tctcccaaca tccttgccag ctttgcaggg 1380 aagaacagagtatgggtcat ctcagcccct catgcctcgg aaggctacta ccgcctcatg 1440 atgagcctgctgaaggacga tgtgtactgt gagctggcgg agaggcacat ccaacagatt 1500 gtgctcttccaccaggcagg tgaggaagga ggcaaggtga gaaggatcac cagcgagggc 1560 cagatcctggagcagcccct ggaccctagc ctcatcccta agctgatgag cttcctgaag 1620 ctggagaagggcaagtttgg catggtgctg ctgaagaaga cgctgcaggt ggaggagcgc 1680 tatccatatcccgttaggct ggaagccatg tacgaggtca tcgaccaagg ccccatccgt 1740 aggatcgagaagatcaggca gaagggcttt gtccagaaat gtaaggcctc tggtgtagag 1800 ggccaggtggtggcggaggg gaatgacggt ggagggggag caggaaggcc aagcctgggc 1860 agcgagaagaagaaagagga cccaaggaga gcacaagtcc caccaaccag agagagtcgg 1920 gtgaaggtcctgagaaaact ggccgccact gcaccagctt tgccccaacc tccctcaacc 1980 cccagagccaccacccttcc tcctgcccca gccacaacag tgactcggtc cacgtcccgg 2040 gcggtaacagttgctgcaag acctatgacc accactgcct ttcccaccac gcagaggccc 2100 tggaccccctcaccctccca caggccccct acaaccactg aggtgatcac tgccaggaga 2160 ccctcagtttcagagaatct ttaccctcca tcccggaagg atcagcacag ggagaggcca 2220 cagacaaccaggaggcccag caaggccacc agcttggaga gcttcacaaa tgcccctccc 2280 accaccatctcagaacccag cacaagggct gctggcccag gccgtttccg ggacaaccgc 2340 atggacaggcgggaacatgg ccaccgagac ccaaatgtgg tgccaggtcc tcccaagcca 2400 gcaaaggagaaacctcccaa aaagaaggcc caggacaaaa ttcttagtaa tgagtatgag 2460 gagaagtatgacctcagccg gcctactgcc tctcagctgg aggacgagct gcaggtgggg 2520 aatgttccccttaaaaaagc aaaggagtct aaaaagcatg aaaagcttga gaaaccagag 2580 aaggagaagaaaaaaaagat gaagaatgag aacgcagaca agttacttaa gagtgaaaag 2640 caaatgaagaagtctgagaa aaagagcaag caagagaaag agaagagcaa gaagaaaaaa 2700 ggaggtaaaacagaacagga tggctatcag aaacccacca acaaacactt cacgcagagt 2760 cccaagaagtcagtggccga cctgctgggg tcctttgaag gcaaacgaag actccttctg 2820 atcactgctcccaaggctga gaacaatatg tatgtgcaac aacgtgatga atatctggaa 2880 agtttctgcaagatggctac caggaaaatc tctgtgatca ccatcttcgg ccctgtcaac 2940 aacagcaccatgaaaatcga ccactttcag ctagataatg agaagcccat gcgagtggtg 3000 gatgatgaagacttggtaga ccagcgtctc atcagcgagc tgaggaaaga gtacggaatg 3060 acctacaatgacttcttcat ggtgctaaca gatgtggatc tgagagtcaa gcaatactat 3120 gaggtaccaataacaatgaa gtctgtgttt gatctgatcg atactttcca gtcccgaatc 3180 aaagatatggagaagcagaa gaaggagggc attgtttgca aagaggacaa aaagcagtcc 3240 ctggagaacttcctatccag gttccggtgg aggaggaggt tgctggtgat ctctgctcct 3300 aacgatgaagactgggccta ttcacagcag ctctctgccc tcagtggtca ggcgtgcaat 3360 tttggtctgcgccacataac cattctgaag cttttaggcg ttggagagga agttggggga 3420 gtgttagaactgttcccaat taatgggagc tctgttgttg agcgagaaga cgtaccagcc 3480 catttggtgaaagacattcg taactatttt caagtgagcc cggagtactt ctccatgctt 3540 ctagtcggaaaagacggaaa tgtcaaatcc tggtatcctt ccccaatgtg gtccatggtg 3600 attgtgtacgatttaattga ttcgatgcaa cttcggagac aggaaatggc gattcagcag 3660 tcactggggatgcgctgccc agaagatgag tatgcaggct atggttacca tagttaccac 3720 caaggataccaggatggtta ccaggatgac taccgtcatc atgagagtta tcaccatgga 3780 tacccttactgagcagaaat atgtaacctt agactcagcc agtttcctct gcagctgcta 3840 aaactacatgtggccagctc cattcttcca cactgcgtac tacatttcct gcctttttct 3900 ttcagtgtttttctaagact aaataaatag caaactttca cctattcatg agttattatt 3960 gaaacctcaaatcataaaga catttaaaag aattgttttt ctaactggag gggctctagt 4020 gctaaataatagtactgaaa attgatatta ttttcctttt cttatatgaa ggaccttatt 4080 tggcatataaaattttataa aatatgtaaa aaaaaaa 4117 7 20 DNA Artificial SequenceDescription of Artificial Sequence Primer 7 taatacgact cactataggg 20 817 DNA Artificial Sequence Description of Artificial Sequence Primer 8atttaggtga cactata 17 9 31 DNA Artificial Sequence Description ofArtificial Sequence Primer 9 tttcacacag gaaacaggaa acagctatga c 31 10 24DNA Artificial Sequence Description of Artificial Sequence Primer 10cgccagggtt ttcccagtca cgac 24 11 20 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 11 ggctaattcg ggagatagcc 20 12 20 DNAArtificial Sequence Description of Artificial Sequence Primer 12caacacctca tggcaagtcc 20 13 28 DNA Artificial sequence Description ofArtificial Sequence Primer 13 cctaaatcta gagcgtcgac gatgctgg 28 14 23DNA Artificial sequence Description of Artificial Sequence Primer 14aagctgttag tcgacccttc aca 23

1. A use of a polynucleotide differentially expressed in ruptured andstable atherosclerotic plaques as a marker for atherosclerosis whereinthe polynucleotide is encoding an amino acid sequence selected from SEQID NO:1, SEQ ID NO:3 or SEQ ID NO:
 5. 2. A method for determining thepresence of a polynucleotide in a sample comprising: obtainingpolynucleotides from an individual and determining whether apolynucleotide encoding an amino acid sequence selected from SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO: 5 is present.
 3. The method according toclaim 2 where the polynucleotide is a RNA polynucleotide.
 4. The methodaccording to claim 2 or 3 comprising the steps of: hybridizing to asample a probe specific for the polynucleotide encoding an amino acidsequence selected from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 underconditions effective for said probe to hybridize specifically to saidpolynucleotide and determining the hybridization of said probe topolynucleotides in said sample.
 5. A method for detecting in a sample aprotein with amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO: 5, said method comprising: incubating with a sample areagent that binds specifically to said protein under conditionseffective for specific binding and determining the binding of saidreagent to said protein in said sample.
 6. A diagnostic processcomprising: determining the difference in expression level of apolynucleotide encoding an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO: 3 or SEQ ID NO: 5 in a sample derived from a host whencompared to a known standard.
 7. A diagnostic process comprising:analyzing for the presence of a protein with amino acid sequenceselected from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 in a samplederived from a host.
 8. An isolated polynucleotide comprising a nucleicacid sequence encoding the amino acid sequence SEQ ID NO:
 1. 9. Thepolynucleotide according to claim 8 comprising the nucleic acid sequenceSEQ ID NO:2.
 10. The polynucleotide according to claim 8 or 9 comprisingthe nucleotides 1169 to 2587 of SEQ ID NO:
 2. 11. The polynucleotideaccording to any one of claims 8-10 consisting of a nucleic acidsequence encoding the amino acid sequence SEQ ID NO:
 1. 12. Thepolynucleotide according to any one of claims 8-11 consisting of thenucleic acid sequence SEQ ID NO:
 2. 3